Inverted chimeric and hybrid oligonucleotides

ABSTRACT

The invention relates to modified oligonucleotides that are useful for studies of gene expression and for the antisense therapeutic approach. The invention provides inverted hybrid oligonucleotides and inverted chimeric oligonucleotides, both of which produce reduced side effects, relative to traditional phosphorothioate, hybrid or chimeric oligonucleotides.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to modified oligonucleotides that are useful forstudies of gene expression and for the antisense therapeutic approach.

2. Summary of the Related Art

The potential for using oligonucleotides as inhibitors of specific geneexpression in an antisense therapeutic approach was first suggested inthree articles published in 1977 and 1978. Paterson et al., Proc. Natl.Acad. Sci. U.S.A. 74: 4370-4374 (1977) discloses that cell-freetranslation of mRNA can be inhibited by binding a complementaryoligonucleotide to the mRNA. Zamecnik and Stephenson, Proc. Natl. Acad.Sci. U.S.A. 75: 280-284 and 285-288 (1978) disclose that a 13-mersynthetic oligonucleotide that is complementary to a part of the Roussarcoma virus (RSV) genome can inhibit RSV replication in infected cellcultures and can inhibit RSV-mediated transformation of primary chickfibroblasts into malignant sarcoma cells.

Since these early studies, the ability of antisense oligonucleotides toinhibit virus propagation has become firmly established. U.S. Pat. No.4,806,463 teaches that human immunodeficiency virus propagation can beinhibited by oligonucleotides that are complementary to any of variousregions of the HIV genome. U.S. Pat. No. 5,194,428 discloses inhibitionof influenza virus replication by phosphorothioate oligonucleotidescomplementary to the influenza virus polymerase 1 gene. Agrawal, Trendsin Biotechnology 10: 152-158 (1992) reviews the use of antisenseoligonucleotides as antiviral agents.

Antisense oligonucleotides have also been developed as anti-parasiticagents. PCT publication no. W093/13740 discloses the use of antisenseoligonucleotides to inhibit propagation of drug-resistant malarialparasites. Tao et al., Antisense Research and Development 5: 123-129(1995) teaches inhibition of propagation of a schistosome parasite byantisense oligonucleotides.

More recently, antisense oligonucleotides have shown promise ascandidates for therapeutic applications for diseases resulting fromexpression of cellular genes. PCT publication no. W095/09236 disclosesreversal of beta amyloid-induced neuronal cell line morphologicalabnormalities by oligonucleotides that inhibit beta amyloid expression.PCT publication no. W094/26887 discloses reversal of aberrant splicingof a globin gene transcript by oligonucleotides complementary to certainportions of that transcript. PCT application no. PCT/US94/13685discloses inhibition of tumorigenicity by oligonucleotides complementaryto the gene encoding DNA methyltransferase.

The development of various antisense oligonucleotides as therapeutic anddiagnostic agents has recently been reviewed by Agrawal and Iyer,Current Opinion in Biotechnology 6: 12-19 (1995).

As interest in the antisense therapeutic approach has grown, variousefforts have been made to improve the pharmacologic properties ofoligonucleotides by modifying the sugar-phosphate backbone. U.S. Pat.No. 5,149,797 describes traditional chimetic oligonucleotides having aphosphorothioate core region interposed between methylphosphonate orphosphoramidate flanking regions. PCT publication no. W094/02498discloses traditional hybrid oligonucleotides having regions of2'-O-substituted ribonucleotides flanking a DNA core region.

Much is currently being discovered about the pharmacodynamic propertiesof oligonucleotides. Agrawal et al., Clinical Pharmacokinetics 28: 7-16(1995) and Zhang et al., Clinical Pharmacology and Therapeutics 58:44-53 (1995) disclose pharmacokinetics of anti-HIV oligonucleotides inhuman patients. Some of these new discoveries have led to new challengesto be overcome for the optimization of oligonucleotides as therapeuticagents. For example, Kniep et al., Nature 374: 546-549 (1995) disclosesthat oligonucleotides containing the CG dinucleotide flanked by certainother sequences have a mitogenic effect in vivo. Galbraith et al.,Antisense Research and Development 4: 201-206 (1994) disclose complementactivation by oligonucleotides. Henry et al., Pharm. Res. 11: PPDM8082(1994) discloses that oligonucleotides may potentially interfere withblood clotting.

There is, therefor, a need for modified oligonucleotides that retaingene expression inhibition properties while producing fewer side effectsthan conventional oligonucleotides.

BRIEF SUMMARY OF THE INVENTION

The invention relates to modified oligonucleotides that are useful forstudies of gene expression and for the antisense therapeutic approach.The invention provides modified oligonucleotides that inhibit geneexpression and that produce fewer side effects than conventionaloligonucleotides. In particular, the invention provides modifiedoligonucleotides that demonstrate reduced mitogenicity, reducedactivation of complement and reduced antithrombotic properties, relativeto conventional oligonucleotides.

In a first aspect, the invention provides inverted hybrid and invertedchimeric oligonucleotides and compositions of matter for inhibitingspecific gene expression with reduced side effects. Such inhibition ofgene expression can be used as an alternative to mutant analysis fordetermining the biological function of specific genes. Such inhibitionof gene expression can also be used to therapeutically treat diseasesthat are caused by expression of the genes of a virus or a pathogen, orby the inappropriate expression of cellular genes.

In one preferred embodiment according to this aspect of the invention,the composition of matter comprises modified oligonucleotides having oneor more 2'-O-substituted RNA region flanked by one or moreoligodeoxyribonucleotide phosphorothioate region. In certainparticularly preferred embodiments, the 2'-substituted RNA region is inbetween two oligodeoxyribonucleotide regions, a structure that is"inverted" relative to traditional hybrid oligonucleotides. In anotherpreferred embodiment according to this aspect of the invention, thecomposition of matter comprises modified oligonucleotides having one ormore nonionic oligonucleotide region flanked by one or more region ofoligonucleotide phosphorothioate. In preferred embodiments, the nonionicregion contains alkylphosphonate and/or phosphoramidate and/orphosphotriester internucleoside linkages. In certain particularlypreferred embodiments, the nonionic oligonucleotide region is in betweentwo oligonucleotide phosphorothioate regions, a structure that is"inverted" relative to traditional chimeric oligonucleotides.

In a second aspect, the invention provides a method for modulating geneexpression in a mammal with reduced side effects. In the methodaccording to this aspect of the invention, a composition of matteraccording to the first aspect of the invention is administered to themammal, wherein the oligonucleotide is complementary to a gene that isbeing expressed in the mammal. In a preferred embodiment, after thecomposition of matter is administered, one or more measurement is takenof biological effects selected from the group consisting of complementactivation, mitogenesis and inhibition of thrombin clot formation.

In a third aspect, the invention provides a method for therapeuticallytreating, with reduced side effects, a disease caused by aberrant geneexpression, the method comprising administering to an individual havingthe disease a composition of matter according to the first aspect of theinvention, wherein the oligonucleotide is complementary to a gene thatis aberrantly expressed, wherein such aberrant expression causes thedisease. In this context, aberrant gene expression means expression in ahost organism of a gene required for the propagation of a virus or aprokaryotic or eukaryotic pathogen, or inappropriate expression of ahost cellular gene. Inappropriate host cellular gene expression includesexpression of a mutant allele of a cellular gene, or underexpression oroverexpression of a normal allele of a cellular gene, such that diseaseresults from such inappropriate host cellular gene expression. In apreferred embodiment, after the composition of matter is administered,one or more measurement is taken of biological effects selected from thegroup consisting of complement activation, mitogenesis and inhibition ofthrombin clot formation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows inverted hybrid oligonucleotides, hybrid oligonucleotidesand oligonucleotide phosphodiesters and phosphorothioates used in thecurrent studies. 2'-O-methylribonucleotides are outlined andphosphodiester-linked nucleotides are underlined; all others arephosphorothioate-linked nucleotides.

FIG. 2 shows mixed backbone, chimeric and inverted chimericoligonucleotides used in the current studies. Methylphosphonate-linkednucleotides are underlined; all others are phosphorothioate linkednucleotides.

FIG. 3 shows thymidine uptake by mouse spleenocytes as a function ofconcentration of phosphorothioate oligonucleotide or any of variousinverted hybrid oligonucleotides.

FIG. 4 shows extent of inhibition of complement-mediated hemolysisobserved when serum is treated with phosphorothioate oligonucleotide orany of various inverted hybrid oligonucleotides.

FIG. 5 shows prolongation of aPTT obtained when normal human serum istreated with phosphorothioate oligonucleotides or with any of variousinverted hybrid oligonucleotides.

FIG. 6 shows thymidine uptake by mouse spleenocytes as a function ofconcentration of phosphorothioate oligonucleotide or any of variousinverted chimeric oligonucleotides.

FIG. 7 shows extent of inhibition of complement-mediated hemolysisobserved when serum is treated with phosphorothioate oligonucleotide orany of various inverted chimetic oligonucleotides.

FIG. 8 shows prolongation of aPTT obtained when normal human serum istreated with phosphorothioate oligonucleotides or with any of variousinverted chimeric oligonucleotides.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All U.S. Patents, patent publications and scientific literature cited inthis specification evidence the level of knowledge in the field and arehereby incorporated by reference.

The invention relates to modified oligonucleotides that are useful forstudies of gene expression and for the antisense therapeutic approach.The invention provides modified oligonucleotides that inhibit geneexpression and that produce fewer side effects than conventionaloligonucleotides. In particular, the invention provides modifiedoligonucleotides that demonstrate reduced mitogenicity, reducedactivation of complement and reduced antithrombotic properties, relativeto conventional oligonucleotides.

In a first aspect, the invention provides inverted hybrid and invertedchimeric oligonucleotides and compositions of matter for inhibitingspecific gene expression with reduced side effects. Such inhibition ofgene expression can be used as an alternative to mutant analysis or gene"knockout" experiments for determining the biological function ofspecific genes. Such inhibition of gene expression can also be used totherapeutically treat diseases that are caused by expression of thegenes of a virus or a pathogen, or by the inappropriate expression ofcellular genes.

A composition of matter for inhibiting specific gene expression withreduced side effects, according to this aspect of the invention,comprises a modified oligonucleotide that is complementary to a portionof a genomic region or gene for which inhibition of expression isdesired, or to RNA transcribed from such a gene. For purposes of theinvention, the term oligonucleotide includes polymers of two or moredeoxyribonucleotide, ribonucleotide, or 2'-O-substituted ribonucleotidemonomers, or any combination thereof. The term oligonucleotide alsoencompasses such polymers having chemically modified bases or sugarsand/or having additional substituents, including without limitationlipophilic groups, intercalating agents, diamines and adamantane.Preferably, such oligonucleotides will have from about 12 to about 50nucleotides, most preferably from about 17 to about 35 nucleotides. Theterm complementary means having the ability to hybridize to a genomicregion, a gene, or an RNA transcript thereof under physiologicalconditions. Such hybridization is ordinarily the result of base-specifichydrogen bonding between complementary strands, preferably to formWatson-Crick or Hoogsteen base pairs, although other modes of hydrogenbonding, as well as base stacking can also lead to hybridization. As apractical matter, such hybridization can be inferred from theobservation of specific gene expression inhibition. The gene sequence orRNA transcript sequence to which the modified oligonucleotide sequenceis complementary will depend upon the biological effect that is soughtto be modified. In some cases, the genomic region, gene, or RNAtranscript thereof may be from a virus. Preferred viruses include,without limitation, human immunodeficiency virus (type 1 or 2),influenza virus, herpes simplex virus (type 1 or 2), Epstein-Barr virus,cytomegalovirus, respiratory syncytial virus, influenza virus, hepatitisB virus, hepatitis C virus and papilloma virus. In other cases, thegenomic region, gene, or RNA transcript thereof may be from endogenousmammalian (including human) chromosomal DNA. Preferred examples of suchgenomic regions, genes or RNA transcripts thereof include, withoutlimitation, sequences encoding vascular endothelial growth factor(VEGF), beta amyloid, DNA methyltransferase, protein kinase A, ApoE4protein, p-glycoprotein, c-MYC protein, BCL-2 protein and CAPL. In yetother cases, the genomic region, gene, or RNA transcript thereof may befrom a eukaryotic or prokaryotic pathogen including, without limitation,Plasmodium falciparum, Plasmodium malarie, Plasmodium ovale, Schistosomaspp., and Mycobacterium tuberculosis.

In addition to the modified oligonucleotide according to the invention,the composition of matter for inhibiting gene expression with reducedside effects may optionally contain any of the well knownpharmaceutically acceptable carriers or diluents. This composition ofmatter may further contain one or more additional oligonucleotidesaccording to the invention, which additional oligonucleotide may beeither an inverted hybrid oligonucleotide or an inverted chimericoligonucleotide. Alternatively, this composition may contain one or moretraditional antisense oligonucleotide, such as an oligonucleotidephosphorthioate, a hybrid oligonucleotide, or a chimericoligonucleotide, or it may contain any other pharmacologically activeagent.

In one preferred embodiment according to this aspect of the invention,the composition of matter comprises modified oligonucleotides having oneor more 2'-O-substituted RNA region flanked by one or moreoligodeoxyribonucleotide phosphorothioate region. In certainparticularly preferred embodiments, the 2'-O-substituted RNA region isin between two oligodeoxyribonucleotide phosphorothioate regions, astructure that is "inverted" relative to traditional hybridoligonucleotides. Accordingly, oligonucleotides according to thisembodiment are designated inverted hybrid oligonucleotides. The2'-O-substituted RNA region preferably has from about four to about 10or 13 2'-O-substituted nucleosides joined to each other by 5' to 3'internucleoside linkages, and most preferably from about four to abouteight such 2'-O-substituted nucleosides. Preferably, the overall size ofthe inverted hybrid oligonucleotide will be from about 15 to about 35 or50 nucleotides. Most preferably, the 2'-O-substituted ribonucleosideswill be linked to each other through a 5' to 3' phosphorothioate,phosphotriester, or phosphodiester linkage. For purposes of theinvention the term "2'-O-substituted" means substitution of the 2'position of the pentose moiety with an -O-lower alkyl group containing1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allylgroup having 2-6 carbon atoms, wherein such alkyl, aryl or allyl groupmay be unsubstituted or may be substituted, e.g., with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halogroup, but not with a 2'-H group. The phosphorothioate flanking regionor regions has from about four to about 46 nucleosides joined to eachother by 5' to 3' phosphorothioate linkages, and preferably from about 5to about 26 such phosphorothioate-linked nucleosides. Most preferably,the phosphorothioate regions will have from about 5 to about 15phosphorothioate-linked nucleosides. The phosphorothioate linkages maybe mixed R_(p) and S_(p) enantiomers, or they may be stereoregular orsubstantially stereoregular in either R_(p) or S_(p) form (see Iyer etal., Tetrahedron Asymmetry 6: 1051-1054 (1995)).

In another preferred embodiment according to this aspect of theinvention, the composition of matter comprises modified oligonucleotideshaving one or more nonionic oligonucleotide region flanked by one ormore region of oligonucleotide phosphorothioate. In preferredembodiments, the nonionic region contains alkylphosphonate and/orphosphoramidate and/or phosphotriester internucleoside linkages. Incertain particularly preferred embodiments, the nonionic oligonucleotideregion is in between two oligonucleotide phosphorothioate regions, astructure that is "inverted" relative to traditional chimericoligonucleotides. Accordingly, oligonucleotides according to thisembodiment are designated inverted chimetic oligonucleotides. Thenonionic region has from about four to about 10 or 12 nucleosides joinedto each other by 5' to 3' nonionic linkages, preferablyalkylphosphonate, phosphoramidate or phosphotriester linkages, andpreferably from about four to about eight such nonionic-linkednucleosides. The phosphorothioate flanking region or regions has fromabout four to about 46 nucleosides joined to each other by 5' to 3'phosphorothioate linkages, and preferably from about eight to about 26such phosphorothioate-linked nucleosides. Most preferably, thephosphorothioate regions will have from about 5 to about 15phosphorothioate-linked nucleosides. The phosphorothioate linkages maybe mixed R_(p) and S_(p) enantiomers, or they may be stereoregular orsubstantially stereoregular in either R_(p) or S_(p) form (see Iyer etal., Tetrahedron Asymmetry 6: 1051-1054 (1995). In a most preferredembodiment the oligonucleotide has a nonionic region having from about 6to about 8 methylphosphonate-linked nucleosides, flanked on either sideby phosphorothioate regions, each having from about 6 to about 10phosphorothioate-linked nucleosides.

Those skilled in the art will recognize that the elements of thesepreferred embodiments can be combined and the inventor does contemplatesuch combination. For example, 2'-O-substituted ribonucleotide regionsmay well include from one to all nonionic internucleoside linkages.Alternatively, nonionic regions may have from one to all2'-O-substituted ribonucleotides. Moreover, oligonucleotides accordingto the invention may contain combinations of one or more2'-O-substituted ribonucleotide region and one or more nonionic region,either or both being flanked by phosphorothioate regions. (SeeNucleosides & Nucleotides 14: 1031-1035 (1995) for relevant synthetictechniques.

In a second aspect, the invention provides a method for modulating geneexpression in a mammal with reduced side effects. In the methodaccording to this aspect of the invention, a composition of matteraccording to the first aspect of the invention is administered to themammal, wherein the oligonucleotide is complementary to a gene that isbeing expressed in the mammal. Preferably, such administration may beparenteral, oral, intranasal or intrarectal. In a preferred embodiment,after the composition of matter is administered, one or more measurementis taken of biological side effects selected from the group consistingof complement activation, mitogenesis and inhibition of thrombin clotformation.

In a third aspect, the invention provides a method for therapeuticallytreating, with reduced side effects, a disease caused by aberrant geneexpression, the method comprising administering to an individual havingthe disease a composition of matter according to the first aspect of theinvention, wherein the oligonucleotide is complementary to a gene thatis aberrantly expressed, wherein such aberrant expression causes thedisease. In this context, aberrant gene expression means expression in ahost organism of a gene required for the propagation of a virus or aprokaryotic or eukaryotic pathogen, or inappropriate expression of ahost cellular gene. Inappropriate host cellular gene expression includesexpression of a mutant allele of a cellular gene, or underexpression oroverexpression of a normal allele of a cellular gene, such that diseaseresults from such inappropriate host cellular gene expression.Preferably, such administration should be parenteral, oral, sublingual,transdermal, topical, intranasal or intrarectal. Administration of thetherapeutic compositions can be carried out using known procedures atdosages and for periods of time effective to reduce symptoms orsurrogate markers of the disease. When administered systemically, thetherapeutic composition is preferably administered at a sufficientdosage to attain a blood level of oligonucleotide from about 0.01micromolar to about 10 micromolar. For localized administration, muchlower concentrations than this may be effective, and much higherconcentrations may be tolerated. Preferably, a total dosage ofoligonucleotide will range from about 0.1 mg oligonucleotide per patientper day to about 200 mg oligonucleotide per kg body weight per day. Itmay desirable to administer simultaneously, or sequentially atherapeutically effective amount of one or more of the therapeuticcompositions of the invention to an individual as a single treatmentepisode. In a preferred embodiment, after the composition of matter isadministered, one or more measurement is taken of biological effectsselected from the group consisting of complement activation, mitogenesisand inhibition of thrombin clot formation.

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to limit thescope of the invention.

EXAMPLE 1 Synthesis, Deprotection And Purification Of Oligonucleotides

Oligonucleotide phosphorothioates were synthesized using an automatedDNA synthesizer (Model 8700, Biosearch, Bedford, Mass.) using abeta-cyanoethyl phosphoramidite approach on a 10 micromole scale. Togenerate the phosphorothioate linkages, the intermediate phosphitelinkage obtained after each coupling was oxidized using 3H,1,2-benzodithiole-3H-one-1,1-dioxide (See Beaucage, In Protocols forOligonucleotides and Analogs: Synthesis and Properties, Agrawal(editor), Humana Press, Totowa, N.J., pp. 33-62 (1993).) Similarsynthesis was carried out to generate phosphodiester linkages, exceptthat a standard oxidation was carried out using standard iodine reagent.Synthesis of inverted chimetic oligonucleotide was carried out in thesame manner, except that methylphosphonate linkages were assembled usingnucleoside methylphosphonamidite (Glen Research, Sterling, Va.),followed by oxidation with 0.1M iodine intetrahydrofuran/2,6-lutidine/water (75:25:0.25) (see Agrawal &Goodchild, Tet. Lett. 28: 3539-3542 (1987). Inverted hybridoligonucleotides were synthesized similarly, except that the segmentcontaining 2'-O-methylribonucleotides was assembled using2'-O-methylribonucleoside phosphoramidite, followed by oxidation to aphosphorothioate or phosphodiester linkage as described above.Deprotection and purification of oligonucleotides was carried outaccording to standard procedures, (See Padmapriya et al., Antisense Res.& Dev. 4: 185-199 (1994)), except for oligonucleotides containingmethylphosphonate-containing regions. For those oligonucleotides, theCPG-bound oligonucleotide was treated with concentrated ammoniumhydroxide for 1 hour at room temperature, and the supernatant wasremoved and evaporated to obtain a pale yellow residue, which was thentreated with a mixture of ethylenediamine/ethanol (1:1 v/v) for 6 hoursat room temperature and dried again under reduced pressure.

EXAMPLE 2 Reduced Complement Activation In Vitro By Inverted Hybrid AndInverted Chimetic Oligonucleotides

To determine the relative effect of inverted hybrid or inverted chimericstructure on oligonucleotide-mediated depletion of complement, thefollowing experiments were performed. Venous blood was collected fromhealthy adult human volunteers. Serum was prepared for hemolyticcomplement assay by collecting blood into vacutainers (Becton Dickinson#6430 Franklin Lakes, N.J.) without commercial additives. Blood wasallowed to clot at room temperature for 30 minutes, chilled on ice for15 minutes, then centrifuged at 4° C. to separate serum. Harvested serumwas kept on ice for same day assay or, alternatively, stored at -70° C.Buffer, oligonucleotide phosphorothioate, inverted hybridoligonucleotide, or inverted chimeric oligonucleotide was then incubatedwith the serum. A standard CH50 assay (see Kabat and Mayer (eds):Experimental Immunochemistry, 2d Edition, Springfield, Ill., C. C.Thomas (1961), p. 125) for complement-mediated lysis of sheep red bloodcells (Colorado Serum Co.) sensitized with anti-sheep red cell antibody(hemolysin, Diamedix, Miami, Fla.) was performed, using duplicatedeterminations of at least five dilutions of each test serum, thenhemoglobin release into cell-free supernates was measuredspectrophotometrically at 541 nm.

EXAMPLE 3 Reduced Mitogenicity In Vitro Of Inverted Hybrid And InvertedChimetic Oligonucleotides

To determine the relative effect of inverted hybrid or inverted chimericstructure on oligonucleotide-mediated mitogenicity, the followingexperiments were performed. Spleen was taken from a male CD1 mouse (4-5weeks, 20-22 g; Charles River, Wilmington, Mass.). Single cellsuspensions were prepared by gently mincing with frosted edges of glassslides. Cells were then cultured in RPMI complete media [RPMI mediasupplemented with 10% fetal bovine serum (FBS), 50 micromolar2-mercaptoethanol (2-ME), 100 U/ml penicillin, 100 micrograms/mlstreptomycin, 2 mM L-glutamine]. To minimize oligonucleotidedegradation, FBS was first heated for 30 minutes at 65° C.(phosphodiester-containing oligonucleotides) or 56° C. (all otheroligonucleotides). Cells were plated in 96 well dishes at 100,000 cellsper well (volume of 100 microliters/well). Oligonucleotides in 10microliters TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA) were added toeach well. After 44 hours of culturing at 37° C., one microcurietritiated thymidine (Amersham, Arlington Heights, Ill.) was added in 20microliters RPMI media for a 4 hour pulse labelling. The cells were thenharvested in an automatic cell harvester (Skatron, Sterling, Va.) andthe filters were assessed using a scintillation counter. In controlexperiments for mitogenicity, cells were treated identically, exceptthat either media (negative control) or concanavalin A (positivecontrol) was added to the cells in place of the oligonucleotides. Theresults of these studies are shown in FIG. 1. All of the inverted hybridoligonucleotides proved to be less immunogenic than phosphorothioateoligonucleotides. Inverted hybrid oligonucleotides having phosphodiesterlinkages in the 2'-O-methyl region appeared to be slightly lessimmunogenic than those containing phosphorothioate linkages in thatregion. No significant difference in mitogenicity was observed when the2'-O-methylribonucleotide region was pared down from 13 to 11 or to 9nucleotides. Inverted chimeric oligonucleotides were also generally lessmitogenic than phosphorothioate oligonucleotides. In addition, theseoligonucleotides appeared to be less mitogenic than traditional chimeticoligonucleotides, at least in cases in which the traditional chimericoligonucleotides had significant numbers of methylphosphonate linkagesnear the 3' end. Increasing the number of methylphosphonate linkers inthe middle of the oligonucleotide from 5 to 6 or 7 did not appear tohave a significant effect on mitogenicity. These results indicate thatincorporation of inverted hybrid or inverted chimeric structure into anoligonucleotide can reduce its mitogenicity.

EXAMPLE 4 Reduced Inhibition Of Clotting In Vitro By Inverted Hybrid AndInverted Chimeric Oligonucleotides

To determine the relative effect of inverted hybrid or inverted chimericstructure on oligonucleotide-induced mitogenicity, the followingexperiments were performed. Venous blood was collected from healthyadult human volunteers. Plasma for clotting time assay was prepared bycollecting blood into siliconized vacutainers with sodium citrate(Becton Dickinson #367705), followed by two centifugations at 4° C. toprepare platelet-poor plasma. Plasma aliquots were kept on ice, spikedwith various test compounds, and either tested immediately or quicklyfrozen on dry ice for subsequent storage at -20° C. prior to coagulationassay. Activated partial thromboplastin time (aPTT) was performed induplicate on an Electra 1000 C (Medical Laboratory Automation, MountVernon, N.Y.) according to the manufacturer's recommended procedures,using Actin FSL (Baxter Dade, Miami, Fla.) and calcium to initiate clotformation, which was measured photometrically. Prolongation of aPTT wastaken as an indication of clotting inhibition side effect produced bythe oligonucleotide. The results are shown in FIG. 5 for inverted hybridoligonucleotides and in FIG. 8 for inverted chimeric oligonucleotides.Traditional phosphorothioate oligonucleotides produce the greatestprolongation of aPTT, of all of the oligonucleotides tested. Traditionalhybrid oligonucleotides produced somewhat reduced prolongation of aPTT.In comparison with traditional phosphorothioate or traditional hybridoligonucleotides, all of the inverted hybrid oligonucleotides testedproduced significantly reduced prolongation of aPTT. Inverted hybridoligonucleotides having phosphodiester linkages in the 2'-O-substitutedribonucleotide region had the greatest reduction in this side effect,with one such oligonucleotide having a 2'-O-methyl RNA phosphodiesterregion of 13 nucleotides showing very little prolongation of aPTT, evenat oligonucleotide concentrations as high as 100 micrograms/ml.Traditional chimeric oligonucleotides produce much less prolongation ofaPTT than do traditional phosphorothioate oligonucleotides. Generally,inverted chimeric oligonucleotides retain this characteristic. At leastone inverted chimetic oligonucleotide, having a methylphosphonate regionof 7 nucleotides flanked by phosphorothioate regions of 9 nucleotides,gave better results in this assay than the traditional chimericoligonucleotides at all but the highest oligonucleotide concentrationstested. These results indicate that inverted hybrid and invertedchimeric oligonucleotides may provide advantages in reducing the sideeffect of clotting inhibition when they are administered to modulategene expression in vivo.

EXAMPLE 5 Reduced Complement Activation In Vivo By Inverted Hybrid AndInverted Chimeric Oligonucleotides

Rhesus monkeys (4-9 kg body weight) are acclimatized to laboratoryconditions for at least 7 days prior to the study. On the day of thestudy, each animal is lightly sedated with ketamine-HCl (10 mg/kg) anddiazepam (0.5 mg/kg). Surgical level anasthesia is induced andmaintained by continuous ketamine intravenous drip throughout theprocedure. Phosphorothioate oligonucleotide or inverted hybrid orinverted chimeric oligonucleotide is dissolved in normal saline andinfused intravenously via a cephalic vein catheter, using a programmableinfusion pump at a delivery rate of 0.42 ml/minute. For eacholigonucleotide, oligonucleotide doses of 0, 0.5, 1, 2, 5 and 10 mg/kgare administered to two animals each over a 10 minute infusion period.Arterial blood samples are collected 10 minutes prior to oligonucleotideadministration and 2, 5, 10, 20, 40 and 60 minutes after the start ofthe infusion, as well as 24 hours later. Serum is used for determiningcomplement CH50, using the conventional complement-dependent lysis ofsheep ertyhrocyte procedure (see Kabat and Mayer, 1961, supra). At thehighest dose, phosphorothioate oligonucleotide causes a decrease inserum complement CH50 beginning within 5 minutes of the start ofinfusion. Inverted hybrid and chimeric oligonucleotides are expected toshow a much reduced or undetectable decrease in serum complement CH50under these conditions.

EXAMPLE 6 Reduced Mitogenicity In Vivo Of Inverted Hybrid And InvertedChimeric Oligonucleotides

CD1 mice are injected intraperitoneally with a dose of 50 mg/kg bodyweight of phosphorothioate oligonucleotide, inverted hybridoligonucleotide or inverted chimeric oligonucleotide. Forty-eight hourslater, the animals are euthanized and the spleens are removed andweighed. Animals treated with inverted hybrid or inverted hybridoligonucleotides are expected to show no significant increase in spleenweight, while those treated with oligonucleotide phosphorothioates areexpected to show modest increases in spleen weight.

EXAMPLE 7 Reduced Inhibition Of Clotting In Vivo By Inverted Hybrid Andinverted Chimetic Oligonucleotides

Rhesus monkeys are treated as in example 5. From the whole blood samplestaken, plasma for clotting assay is prepared, and the assay performed,as described in example 4. It is expected that prolongation of aPTT willbe substantially reduced for both inverted hybrid oligonucleotides andfor inverted chimetic oligonucleotides, relative to traditionaloligonucleotide phosphorothioates.

EXAMPLE 8 Effect Of Inverted Hybrid Or Chimeric Structure On RNase HActivity

To determine the ability of inverted hybrid oligonucleotides andinverted chimetic oligonucleotides to actrate RNase H when bound to acomplementary RNA molecule, the following experiments were performed.Each oligonucleotide phosphorothioate, inverted hybrid oligonucleotideor inverted chimeric oligonucleotide was incubated together with a molarequivalent quantity of complimentary oligoribonucleotide (0.266micromolar concentration of each), in a cuvette containing a finalvolume of 1 ml RNase H buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 0.1MKCl, 2% glycerol, 0.1 mM DTT). The samples were heated to 95° C., thencooled gradually to room temperature to allow annealing to form duplexeshealed duplexes were incubated for 10 minutes at 37° C., then 5 unitsRNase H was added and data collection commenced over a three hourperiod. Data was collected using a GBC 920 (GBC Scientific Equipment,Victoria, Australia) spectrophotometer at 259 nm. RNase H degradationwas determined by hyperchromic shift.

The results are shown in Table I, below.

                  TABLE I    ______________________________________    RNase H Degradation Of Oligonucleotides    Oligo No. (Features)                Half-Life Oligo No. (Features)                                        Half-Life    ______________________________________    GEM91 (all PO)                 8.8 sec  Hyb115 (5' MP)                                        11.5 sec.    GEM91 (all PS)                22.4 sec. Hyb116 (chimeric)                                         9.7 sec.    GEM91H (hybrid)                32.7 sec. Hyb117 (chimeric)                                         8.1 sec.    Hyb108 (inv. hyb.)                15.4 sec. Hyb118 (inv. chim.)                                        11.5 sec.    Hyb109 (inv. hyb.)                 7.9 sec. Hyb119 (inv. chim.)                                        14.4 sec.    Hyb110 (inv. hyb.)                10.4 sec. Hyb120 (inv. chim.)                                         9.3 sec.    Hyb111 (inv. hyb.)                12.9 sec. Hyb121 (3' MP)                                        21.2 sec.    Hyb112 (inv. hyb.)                12.5 sec. Hyb122 (chimeric)                                        23.0 sec.    Hyb113 (inv. hyb.)                10.9 sec. Hyb123 (chimeric)                                        41.8 sec.    Hyb114 (inv. hyb.)                20.3 sec. Hyb124 (chimeric)                                        not detect.    ______________________________________

As expected, phosphodiester oligonucleotides behaved as very goodco-substrates for RNase H-mediated degradation of RNA, with adegradative half-life of 8.8 seconds. Phosphorothioate oligonucleotidesproduced an increased half-life of 22.4 seconds. Introduction of a2'-O-methylribonucleotide segment at either end of the oligonucleotidefurther worsened RNaseH activity (half-life=32.7 seconds). In contrast,introducing a 2'-O-methyl segment into the middle of the oligonucleotide(inverted hybrid structure) always resulted in improved RNase H-mediateddegradation. When a region of 13 2'-O-methylribonucleosidephosphodiesters was flanked on both sides by phosphorothioate DNA, thebest RNase H activity was observed, with a half-life of 7.9 seconds.Introduction of large blocks of methylphosphonate-linked nucleosides atthe 3' end of the oligonucleotide either had no effect or caused furtherdeterioration of RNase H activity even when in a chimeric configuration.Introduction of methylphosphonate linked nucleosides at the 5' end,however, improved RNase H activity, particularly in a chimericconfiguration with a single methylphosphonate linker at the 3' end (besthalf-life=8.1 seconds). All inverted chimetic oligonucleotides withmethylphosphonate core regions flanked by phosphorothioate regions gavegood RNase results, with a half-life range of 9.3 to 14.4 seconds. Theseresults indicate that the introduction of inverted hybrid or invertedchimeric structure into phosphorothioate-containing oligonucleotides canrestore some or all of the ability of the oligonucleotide to act as aco-substrate for RNase H, a potentially important attribute for aneffective antisense agent.

EXAMPLE 9 Effect Of Inverted Hybrid Or Chimetic Structure On MeltingTemperature

To determine the effect of inverted hybrid or inverted chimeticstructure on stabilty of the duplex formed between an antisenseoligonucleotide and a target molecule, the following experiments wereperformed. Thermal melting (Tm) data were collected using a GBC 920spectrophotometer, which has six 10 mm cuvettes mounted in a dualcarousel. In the Tm experiments, the temperature was directed andcontrolled through a peltier effect temperature controller by acomputer, using software provided by GBC, according to themanufacturer's directions. Tm data were analyzed by both the firstderivative method and the mid-point method, as performed by thesoftware. Tm experiments were performed in a buffer containing 10 mMPIPES, pH 7.0, 1 mM EDTA, 1M NaCl. A VWR 1166 (VWR, Boston, Mass.)refrigerated bath was connected to the peltier-effect temperaturecontroller to absorb the heat. Oligonucleotide strand concentration wasdetermined using absorbance values at 260 nm, taking into accountextinction coefficients.

The results are shown in Table II, below.

                  TABLE II    ______________________________________    Duplex Stability Of Oligonucleotides    Oligo No. (Features)                Tm (°C.)*                          Oligo No. (Features)                                        Tm (°C.)*    ______________________________________    GEM91 (all PO)                72.0      Hyb115 (5' MP)                                        61.8    GEM91 (all PS)                63.6      Hyb116 (chimeric)                                        61.0    GEM91H (hybrid)                67.0      Hyb117 (chimeric)                                        60.5    Hyb108 (inv. hyb.)                76.4      Hyb118 (inv. chim.)                                        57.9    Hyb109 (inv. hyb.)                80.0      Hyb119 (inv. chim.)                                        57.7    Hyb110 (inv. hyb.)                74.2      Hyb120 (inv. chim.)                                        56.8    Hyb111 (inv. hyb.)                76.9      Hyb121 (3' MP)                                        60.7    Hyb112 (inv. hyb.)                72.1      Hyb122 (chimeric)                                        60.5    Hyb113 (inv. hyb.)                74.3      Hyb123 (chimeric)                                        59.0    Hyb114 (inv. hyb.)                71.3      Hyb124 (chimeric)                                        not detect.    ______________________________________     *= with complementary RNA

These results demonstrate that conversion of a phosphodiesteroligonucleotide to a phosphorothioate oligonucleotide results in areduction of duplex stability, and that introduction ofmethylphosphonate linkages further reduces duplex stability. Duplexstabilty can be restored by adding 2'-O-methylribonucleotides, and canexceed that of the phosphodiester oligonucleotide when an invertedhybrid structure is used. Conversely, use of an inverted chimericstructure results in the lowest melting temperatures observed for anyhybridizing methylphosphonate-containing oligonucleotide, althoughduplex stability was still well above physiological temperatures. Takentogether, these results suggest that inverted hybrid or invertedchimeric structure can be used to custom design oligonucleotides forparticular duplex stabilities desired in particular experimental ortherapeutic applications.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 20    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CTCTCGCACCCAUCUCUCUCCTTCT25    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    CTCTCGCACCCAUCUCUCUCCTTCT25    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 25 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: Other nucleic acid    (xi) SEQUENCE DESCRIPTION: SEQ ID 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What is claimed is:
 1. A modified oligonucleotide having from about 15to about 35 nucleotides, such modified oligonucleotide comprising a2'-O-substituted RNA region in between two oligodeoxyribonucleotidephosphorothioate regions, wherein the 2'-O-substituted RNA region hasfrom about 4 to about 13 phosphodiester-linked 2'-O-substitutedribonucleosides, each of which has a 2'-O-substitution selected from ahydroxy group, an amino group, a halo group and an --O-lower alkyl groupcontaining 1-6 saturated or unsaturated carbon atoms or an --O-aryl orallyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allylgroup may be unsubstituted or may be substituted with halo, hydroxy,trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl,carbalkoxyl, or amino groups; and wherein each of theoligodeoxyribonucleotide phosphorothioate regions has from about 5 toabout 15 phosphorothioate-linked nucleosides.
 2. A composition of matterfor inhibiting gene expression with reduced side effects, thecomposition comprising the modified oligonucleotide according to claim1.